Immunogenic polypeptides encoded by MAGE minigenes and uses thereof

ABSTRACT

The invention discloses immunogenic polypeptides comprising several MAGE-specific antigen epitopes selected from different (i.e. discrete) members of the MAGE protein family, nucleic acids coding therefor, recombinant viruses and/or cells comprising said nucleic acids, and compositions thereof. Methods for eliciting or inducing MAGE-specific immune responses utilizing the aforementioned immunogenic agents are also disclosed.

FIELD OF THE INVENTION

The present invention relates to immunology, in particular to novelpolypeptides comprising several MAGE-specific antigen epitopes thereinselected from different (i.e. discrete) members of the MAGE proteinfamily, nucleic acids coding therefor, recombinant viruses comprisingsaid nucleic acid, cells comprising said nucleic acids, compositionsthereof, and their use as immunogenic agents.

BACKGROUND OF THE INVENTION

The prospects of cancer immunotherapy rely upon the identification oftumor associated antigens which can be recognized by the immune system.Specifically, target antigens eliciting T cell-mediated responses are ofcritical interest. This comes from evidence that cytotoxic T lymphocytes(CTLs) can induce tumor regression both in animal models (Kast W. et al(1989) Cell 59:6035; Greendberg P. (1991) Adv. Immunol. 49:281) and inhumans (Boon T. et al. (1994) Annu. Rev. Immunol. 12:337). Antigensrecognized by CTLs consist of peptides originating from endogenousproteins presented in association with Major Histocompatibility Complex(MHC) molecules.

The first such human tumor associated antigen characterized wasidentified from a melanoma. This antigen (originally designated MAGE 1)was identified using CTLs isolated from an HLA A1+melanoma patient toscreen HLA A1 target cells transfected with tumor DNA (van der BruggenP. (1991) Science 254:1643; said tumor associated antigens are nowdesignated MAGE-A1, MAGE-A2, etc.—for purposes of this application theolder designation will be adopted). Interestingly, MAGE 1 was found tobelong to a family of at least 12 closely related genes located on the Xchromosome (de Plaen, B. et al. (1994) Immunogenetics 40:360). Thenucleic acid sequence of the 11 additional MAGE genes share 65-85%identity with that of MAGE-1 (de Smet, C. et al. (1994) Immunogenetics39: 121). Both MAGE 1 and 3 are present in normal tissues, but expressedonly in the testis (de Plaen, E. et al. (1994) Supra; de Smet, C. et al.(1994) Supra; Takahashi, K. et al. (1995) Cancer Res. 55:3478; Chomey,P. et al. (1995) Immunogenetics 43:97). These initial results havesubsequently been extended with the identification of new gene families(i.e. RAGE, BAGE, GAGE), all of which are typically not expressed innormal tissues (except testis) but expressed in a variety of tumortypes.

MAGE-1 and MAGE-3 are expressed in 48% and 76% of metastatic melanomasrespectively (Brasseur, F. et al. (1995) Int. J. Cancer 63:375), 35% and30% of non-small cell lung cancer (“NSCLC”; Weynants, P. et al. (1994)Int. J. Cancer 56:826), 27% and 48% of head and neck, squamous cellcarcinomas (Eura, M. et al. (1995) Int. J. Cancer 64:304), 62% and 57%of esophageal squamous cell carcinomas (Inoue, H. et al. (1995) Int. J.Cancer 63:523), and 21% and 35% of bladder transitional cell carcinomas(Patard, J.-J. et al. (1995) Int. J. Cancer 64:60). They are alsoexpressed (albeit less frequently) in tumors of other histological typessuch as breast tumors, soft tissue sarcomas and prostatic carcinoma(Brasseur, F. et al. (1992) Int. J. Cancer 52:839; Van der Eynde et al.(1995) Curr. Opin. Immunol. 7:674). The MAGE 3 gene is also expressed incolorectal carcinoma.

A number of immunogenic epitopes/peptides derived from MAGE proteins (inparticular MAGE 1, 2 and 3) have been identified and characterized(reviewed in van der Eynde, B. J. and Boon, T. (1997) Int. J. Chem. LabRes. 27:81). CTLs isolated from two melanoma patients were found torecognize MAGE-1 derived peptides presented in association with HLA-A1,B37 or Cw16 (Traversi, C. et al (1992) J. Exp. Med 176:1453; Tanzarella,S. et al. (1999) Cancer Res. 59:2668; van der Bruggen, P. et al. (1994)Eur. J. Immunol. 24:2134). In particular, the nonapeptide EADPTGHSY(amino acids 161-169 of MAGE 1) was demonstrated to be presented tocells of the immune system in association with the MHC class 1 moleculeHLA-A1 (Traversi, C. et al. (1992) Supra). Synthetic peptides utilizedto stimulate T cells have also facilitated the identification of a MAGE1 epitope presented by HLA-A24 (Fujie, T et al. (1999) Int. J. Cancer80:169).

It has also been demonstrated that MAGE-3 directs the expression of anumber of antigens recognized by CTLs. For example, the nonapeptideEVDPIGHLY (amino acid 168-176 of MAGE 3) is recognized in associationwith the MHC class 1 molecule HLA-A1 (Gaugler, B. et al. (1994) J. Exp.Med 179:921); the decapeptide MEVDPIGHLY (amino acids 167-176) inassociation with the MHC class 1 molecules HLA-B44.02 and HLA-B44.03(Herman, J. et al. (1996) Immunogenetics 43:377); the nonapeptideFLWGPRALV (amino acids 271-279) in association with the MHC class 1molecule HLA-A2.01 (van der Bruggen, P. et al. (1994) Eur. J. Immunol24:3038). Furthermore, van der Bruggen and colleagues have identifiedsix additional MAGE-derived epitopes that are presented to CTL inassociation with HLA-A3, A28, B7, B53, Cw2 and Cw3 (Chaux, P. et al.(1999) J. Immunol 163:2928; Luiten, R. et al. (2000), Tissue Antigens,55: in press). Interestingly, it has recently been observed that anepitope of MAGE 1 previously identified as an epitope recognized by CTLin association with HLA-A1 can also be presented to CTL in associationwith both HLA-B3501 and B3503 (van der Bruggen, P. et al. (2000),submitted).

The present invention discloses novel polypeptides comprising severaldistinct MAGE-specific antigen epitopes selected from different (i.e.discrete) members of the MAGE protein family, nucleic acids codingtherefor, recombinant viruses and/or cells comprising said nucleicacids, compositions of the aforementioned agents, and their advantageoususe in generating MAGE-specific immune responses.

SUMMARY OF THE INVENTION

The present invention encompasses immunogenic peptides comprisingseveral MAGE-specific antigen epitopes selected from different (i.e.discrete) members of the MAGE protein family, nucleic acids codingtherefor, recombinant virus and/or cells comprising said nucleic acids,and compositions of the aforementioned. All of these aforementionedagents and compositions are characterized by their ability to induce orelicit an immune response against said polypeptide, a MAGE-specificantigen epitope of said polypeptide, a MAGE protein or fragment thereofcomprising a MAGE-specific antigen epitope of said polypeptide, or cellsbinding and/or expressing the aforementioned polypeptide, MAGE-specificantigen epitope, MAGE protein polypeptide or fragment thereof.

Accordingly, in one aspect of the invention a polypeptide is providedwherein the MAGE-specific antigen epitopes are derived from MAGE 1, MAGE2 and/or MAGE 3. Further aspects of the invention encompass polypeptideswherein said MAGE-specific antigen epitopes are directly adjoinedtogether, or are joined via an amino acid linker sequence.

In a further aspect of the invention, the polypeptide comprises a firstMAGE-specific antigen epitope derived from MAGE 1 having the amino acidsequence EADPTGHSY and a second MAGE-specific antigen epitope derivedfrom MAGE-3 having the amino acid sequence EVDPIGHLY. In yet furtheraspects, the polypeptide consists/comprises the amino acid sequence ofSEQ ID NO:1 (FIG. 2).

As previously noted, aspects of the invention encompass nucleic acidscoding for the aforementioned polypeptides. Accordingly, aspects of theinvention consist/comprise the nucleic acid sequence of SEQ ID NO:2(FIG. 1). In further aspects of the invention, the nucleic acid is a DNAselected from the group consisting of viral nucleic acid, plasmid,bacterial DNA, naked/free DNA, and RNA. In yet further aspects, theviral nucleic acid is selected from the group consisting of adenovirus,alpha-virus and poxvirus. In still yet further embodiments, the poxvirusis selected from the group consisting of ALVAC, NYVAC, TROVAC and MVA.

Particular aspects of the invention further encompass recombinantviruses into which is inserted a nucleic acid encoding for a polypeptideof the invention, wherein the recombinant virus causes the expression ofthe polypeptide in an infected cell. In a further aspect of theinvention, cells infected with said recombinant viruses are capable ofeliciting an immune response directed against:

-   -   (i) the polypeptide; and/or    -   (ii) a MAGE-specific antigen epitope of the polypeptide; and/or    -   (iii) a MAGE protein or fragment thereof comprising a        MAGE-specific antigen epitope of the polypeptide; and/or    -   (iv) cells expressing MAGE protein or fragments thereof, the        polypeptide, a MAGE-specific antigen epitope of the polypeptide;        and/or    -   (v) cells binding said MAGE protein or fragments thereof, the        polypeptide, a MAGE-specific antigen epitope of the polypeptide.

Further aspects of the invention encompass recombinant viruses selectedfrom the group consisting of adenovirus, alphavirus and poxvirus;particular embodiments encompass ALVAC.

Further aspects of the invention encompass compositions of theaforementioned polypeptides, nucleic acids, and recombinant viruses.These compositions may optionally include adjuvants.

The invention further provides for cells comprising the aforementionednucleic acid(s), wherein said cells express a polypeptide of theinvention. In further aspects of the invention, the cells bind cleavagefragments of a polypeptide of the invention (these latter fragmentsoptionally produced by a protease). In yet further aspects, the cellsexpressing the polypeptide also express a MHC HLA class 1 molecule. Instill yet further aspects, the cells expressing the polypeptide areantigen-presenting cells.

The invention further provides a method of inducing an immune responsein an animal directed against:

-   -   (i) the polypeptide; and/or    -   (ii) a MAGE-specific antigen epitope of the polypeptide; and/or    -   (iii) a MAGE protein or fragment thereof comprising a        MAGE-specific antigen epitope of the polypeptide; and/or    -   (iv) cells expressing MAGE protein or fragments thereof, the        polypeptide, a MAGE-specific antigen epitope of the polypeptide;        and/or    -   (v) cells binding said MAGE protein or fragments thereof, the        polypeptide, a MAGE-specific antigen epitope of the polypeptide,        comprising administering to said animal a polypeptide, or a        nucleic acid, or a recombinant virus, or a cell of the invention        in an amount sufficient to induce an immune response.

The invention in yet a further aspect provides for a treatment forcancer comprising any one of the aforementioned methods for inducingimmune responses.

The invention in still a further aspect provides for the use of thepolypeptides of the invention in the manufacture of a medicament for thetreatment of cancer.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating particular embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

The invention will be better understood with reference to the drawingsin which:

FIG. 1 shows a single stranded DNA sequence comprising a MAGE 1/3Minigene encoded thereby (SEQ ID NO: 2) corresponding to the insert ofthe ALVAC(1)-MAGE 1/3 Minigene construct designated vCP1469A.

FIG. 2 shows the amino acid sequence (SEQ ID NO:1) of the MAGE 1/3Minigene encoded by the DNA sequence of FIG. 1.

FIG. 3 depicts a schematic representation of the Xho I restriction mapprofile of the ALVAC(1)-MAGE 1/3 Minigene construct (vCP1469A).

FIG. 4 shows data depicting the capacity of the ALVAC(1)-MAGE 1/3Minigene construct (vCP1469A) of FIG. 3 to express both the MAGE 1 and 3specific antigen epitope following infection of dendritic cells withrecombinant virus comprising said ALVAC construct.

FIG. 5 shows data depicting the capacity of the MAGE 1 specific antigenepitope derived from the ALVAC(1)-MAGE 1/3 Minigene construct (vCP1469A)of FIG. 3 to present itself in association with both MHC HLA-A1 andHLA-B35 molecules.

FIG. 6 shows data depicting the capacity of the MAGE 3 specific antigenepitope derived from the ALVAC(1)-MAGE1/3 Minigene construct (vCP1469A)of FIG. 3 to present itself in association with MHC HLA-B35 molecules.

DETAILED DESCRIPTION OF THE INVENTION

The invention, discloses immunogenic polypeptides comprising severalMAGE-specific antigen epitopes selected from different (i.e. discrete)members of the MAGE protein family, nucleic acids coding therefor,recombinant viruses and/or cells comprising said nucleic acids(collectively designated as “agents” of the invention), and compositionsof the aforementioned. All of the aforementioned agents and compositionsof the invention have the ability to induce or elicit an immune responseagainst said polypeptide, a MAGE-specific antigen epitope of saidpolypeptide, a MAGE protein or fragment thereof comprising aMAGE-specific antigen epitope of said polypeptide, or cells bindingand/or expressing the aforementioned polypeptide, MAGE-specific antigenepitope, MAGE protein or fragment thereof. An “immune response” isdefined as any response of the immune system, for example, of either acell-mediated (i.e. cytotoxic T-lymphocyte mediated) or humoral (i.e.antibody mediated) nature.

Within the context of cell-mediated immune responses, tumor associatedantigen proteins (such as members of the MAGE family) are processed byintracellular proteases into smaller epitope peptides which aresubsequently transported to the cell surface tightly bound in a cleft onan MHC HLA class 1 molecule. T cells recognize these small epitopepeptides only when presented in association with MHC HLA Class Imolecules on the surface of appropriate cells. Analogously, in thecontext of humoral immune responses proteins can be processed intosmaller epitope peptides which are subsequently presented on cellsurfaces (i.e. antigen presenting cells) in association with MHC HLAclass II molecules. Said complexes are recognized by appropriate cellsof the humoral immune system.

As is well known to those skilled in the art, short peptides (i.e.epitopes) composed of amino acid sequences of about 9 to 12 amino acidsderived from antigens are capable of binding directly within the cleftof an HLA class 1 molecule without intracellular processing. Aspreviously noted, a number of such epitope peptides derived from MAGEproteins (i.e. MAGE-specific antigen epitopes) have been identified.Moreover, some of these MAGE-specific antigen epitopes have demonstratedthe capacity to induce/elicit immune responses wherein appropriateMAGE-expressing target cells are lysed. The polypeptides of the presentinvention elicit an improved immune response to appropriateMAGE-expressing target cells by comparison to that observed when only asingle MAGE-specific antigen epitope is employed as immunogen. Moreover,the appropriate choice of epitopes will provide the advantage of animmunogenic polypeptide capable of eliciting or inducing an immuneresponse in a population of individuals not restricted to a single MHCHLA subclass. In addition, the linking of MAGE-specific antigen epitopesin a single polypeptide allows for the administration of a singleimmunogen to individuals (by contrast to a mixture of distinctindividual peptides). As such, the polypeptide is appropriatelyprocessed to allow the individual MAGE-specific antigen epitopesencompassed therein to be presented in association with the relevant HLAclass 1 molecules on relevant cell surfaces.

The individual MAGE-specific antigen epitopes which comprise thepolypeptide of the present invention may each vary in the number ofamino acids, typically comprising from about 8 to about 12 amino acids,but preferably of the size of 9 to 10 amino acids. In one embodiment ofthe invention, the MAGE-specific antigen epitopes each comprise 9 aminoacids.

Any MAGE-specific antigen epitope derived from any member of the MAGEprotein family can be incorporated into the polypeptides of the presentinvention. For example, a number of MAGE 1-specific antigen epitopes aredisclosed in U.S. Pat. Nos. 5,405,940, 5,695,994, 5,843,448 andpublished PCT application WO 9504542; a number of MAGE 2-specificantigen epitopes are disclosed in U.S. Pat. Nos. 5,554,724, 5,686,068and published PCT application WO 9525530; a number of MAGE 3-specificantigen epitopes are disclosed in U.S. Pat. Nos. 5,462,871, 6,025,474,5,851,523, 5,965,535, 5,551,506, 5,591,430, 5,585,461 and published PCTapplications WO 9525739, WO 9626214 and WO 9731017; and MAGE 6-specificantigen epitopes are disclosed in published PCT application WO 9810780(all of which are herein incorporated by reference).

In one embodiment of the invention, the polypeptide comprises a firstMAGE-specific antigen epitope selected from MAGE 1 and a second chosenfrom MAGE 3. In a further embodiment of the invention, the MAGE-1specific antigen epitope comprises the amino acid sequence EADPTGHSY,The MAGE-3 specific antigen epitope may comprise the amino acid sequenceEVDPIGHLY. It should be noted that the aforementioned epitopesencompassed by the polypeptide embodiments of this invention can bedirectly adjoined together, or be connected by an amino acid linkersequence. Said amino acid linker sequences for joining the MAGE-specificantigen epitopes may comprise from 1 to about 25 ammo acids. In oneembodiment, the linker comprises 16 amino acids. In further embodimentsof the invention, the polypeptide and/or amino acid linker sequencefurther comprises an amino acid sequence cleavable by proteolyticactivity. A particular embodiment of the invention consists of thepolypeptide whose sequence is denoted by SEQ ID NO:1 (FIG. 2).

The polypeptides of the present invention may also encompass“functionally equivalent variants” or “analogs” of the polypeptides. Assuch, this would include but not be limited to polypeptides with partialsequence homology, peptides having one or more specific conservativeand/or non-conservative amino acid changes and peptide conjugates whichdo not alter the biological or structural properties of the polypeptide.

The polypeptides of the invention may be prepared using a variety ofmethods known to one skilled in the art. Accordingly, recombinant DNAmethods can be utilized to provide these polypeptides. Nucleic acidsequences which encode for the polypeptides of the invention may beincorporated in a known manner into appropriate expression vectors (i.e.recombinant expression vectors). Possible expression vectors include(but are not limited to) cosmids, plasmids, or modified viruses (e.g.replication defective retroviruses, adenoviruses and adeno-associatedviruses, lentiviruses, poxviruses), so long as the vector is compatiblewith the host cell used. The expression “vector is compatible with thehost cell” is defined as contemplating that the expression vector(s)contain a nucleic acid molecule of the invention (hereinafter described)and attendant regulatory sequence(s) selected on the basis of the hostcell(s) to be used for expression, said regulatory sequence(s) beingoperatively linked to the nucleic acid molecule. “Operatively linked” isintended to mean that the nucleic acid is linked to regulatorysequence(s) in a manner which allows expression of the nucleic acid.Suitable regulatory sequences may be derived from a variety of sources,including bacterial, fungal, or viral genes. (For example, see theregulatory sequences described in Goeddel, Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).)Selection of appropriate regulatory sequence(s) is dependent on the hostcell(s) chosen, and may be readily accomplished by one of ordinary skillin the art. Examples of such regulatory sequences include the following:a transcriptional promoter and enhancer, RNA polymerase bindingsequence, or a ribosomal binding sequence (including a translationinitiation signal). Depending on the host cell chosen and the vectoremployed, other additional sequences (such as an origin of replication,additional DNA restriction sites, enhancers, and sequences conferringinducibility of transcription) may be incorporated into the expressionvector.

The aforementioned expression vectors of the invention may also containa selectable marker gene which facilitates the selection of host cellstransformed or transfected with a recombinant molecule of the invention.Examples of selectable marker genes are genes encoding a protein such asG418 and hygromycin (which confer resistance to certain drugs),β-galactosidase, chloramphenicol acetyltransferase, or fireflyluciferase.

Transcription of the selectable marker gene is monitored by changes inthe concentration of the selectable marker protein such asβ-galactosidase, chloramphenicol acetyltransferase, or fireflyluciferase. Transformant cells can be selected with appropriateselection molecules if the selectable marker gene encodes a proteinconferring antibiotic resistance (i.e. G418 in context of neomycinresistance). As is known to one skilled in the art, cells that haveincorporated the selectable marker gene will survive, while cells whichdo not have any such incorporated detectable marker will die. This makesit possible to visualize and assay for expression from recombinantexpression vectors of the invention. It will also be appreciated thatselectable markers can be introduced on a separate vector from thenucleic acid of interest.

The recombinant expression vectors may also contain genes which encode afusion moiety which provides increased expression of the polypeptides ofthe invention; increased solubility of the polypeptides of theinvention; and/or aids in the purification of a target recombinantprotein by acting as a ligand in affinity purification. For example, aproteolytic cleavage site may be added to the target recombinantpolypeptide to allow separation of the recombinant polypeptidepeptide(s) from the fusion moiety subsequent to purification of thefusion protein.

The polypeptides of the invention may also be prepared by chemicalsynthesis using techniques well known in the chemistry of proteins suchas solid phase synthesis (Merrifield (1964) J. Am. Chem. Assoc, 85:2149)or synthesis in homogenous solution (Methods of Organic Chemistry, E.Wansch (Ed.) Vol. 15, pts. I and II, Thieme, Stuttgart (1987)).

Additional embodiments of the invention encompass nucleic acids codingfor the polypeptides hereinbefore described. As defined herein, “nucleicacid(s)” encompass (but is not limited to) viral nucleic acid(s),plasmid(s), bacterial DNA, naked/free DNA and RNA. The nucleic acidsencompass both single and double stranded forms. As such, these nucleicacids comprise the relevant base sequences coding for the aforementionedpolypeptides. For purposes of definitiveness, the “relevant basesequences coding for the aforementioned polypeptides” further encompasscomplementary nucleic acid sequences.

In one embodiment of the invention, the nucleic acid has the sequencedenoted by SEQ ID NO:2 (FIG. 1). In further embodiments of theinvention, the nucleic acids comprise this sequence (i.e. SEQ ID NO:2(FIG. 1)).

As stated above, the present invention the present invention alsoencompasses nucleic acid sequences which are complementary as well asanticomplementary to the sequence denoted by SEQ ID NO:2 and equivalentsequence variants thereof. One skilled in the art may determine suchcomplementary or anticomplementary nucleic acid sequences. Also as partof the invention, are nucleic acid sequences which hybridize to one ofthe aforementioned nucleic acid molecules under stringent conditions.“Stringent conditions” as used herein refers to parameters with whichthe art is familiar and such parameters are discussed for example inMolecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989 or Current Protocols in Molecular Biology, F. M. Ausubel, etal., eds., John Wiley & Sons Inc., New York. One skilled in the artwould be able to identify homologs of nucleic acids encoding the MAGEpolypeptides of the invention as well as screening cells and librariesfor expression of such molecules which then are isolated, followed byisolated of the pertinent nucleic acid molecule and sequencing.

It is noted that the nucleic acid molecules described herein represent apreferred embodiment of the invention. The invention also encompassesdegenerate nucleic acids that differ from the aforementioned sequences.Due to degeneracy in the genetic code, variations in the DNA sequencewill result in translation of equivalent peptides. It is thus understoodthat numerous choices of nucleotides may be made that will lead to asequence capable of directing production of the polypeptides orfunctional analogs thereof of the present invention. As a result,substitutions are included in the scope of the invention.

The viral nucleic acid of the invention may be derived from a poxvirusor other virus such as adenovirus or alphavirus. As such, the viralnucleic acids further comprising a nucleic acid sequence coding for apolypeptide of the invention is designated for discussion hereinafter asa “‘viral’ vector”. Preferably the viral vector is incapable ofintegration in recipient animal cells. The elements for expression fromsaid vector may include a promoter suitable for expression in recipientanimal cells.

Adenovirus vectors and methods for their construction have beendescribed (e.g. U.S. Pat. Nos. 5,994,132, 5,932,210, 6,057,158 andPublished PCT Applications WO 9817783, WO 9744475, WO 9961034, WO9950292, WO 9927101, WO 9720575, WO 9640955, WO 9630534—all of which areherein incorporated by reference). Alphavirus vectors have also beendescribed in the art and can be used in embodiments of this invention(e.g. U.S. Pat. Nos. 5,792,462, 5,739,026, 5,843,723, 5,789,245, andPublished PCT Applications WO 9210578, WO 9527044, WO 9531565, WO9815636—all of which are herein incorporated by reference), as havelentivirus vectors (e.g. U.S. Pat. Nos. 6,013,516, 5,994,136 andPublished PCT Applications WO 9817816, WO 9712622, WO 9817815, WO9839463, WO 9846083, WO 9915641, WO 9919501, WO 9930742, WO 9931251, WO9851810, WO 0000600—all of which are herein incorporated by reference).Poxvirus vectors that can be used include, for example, vaccinia andcanary poxvirus (as described in U.S. Pat. Nos. 5,364,773, 4,603,112,5,762,938, 5,378,457, 5,494,807, 5,505,941, 5,756,103, 5,833,975 and5,990,091—all of which are herein incorporated by reference). Poxvirusvectors comprising a nucleic acid coding for polypeptides of theinvention can be obtained by homologous recombination as is known to oneskilled in the art. As such, the polynucleotide of the invention isinserted into the viral genome under appropriate conditions torexpression in mammalian cells (as described below).

In one embodiment of the invention the poxvirus vector is ALVAC (1) orALVAC (2) (both of which have been derived from canarypox virus). ALVAC(1) (or ALVAC (2)) does not productively replicate in non-avian hosts, acharacteristic thought to improve its safety profile. ALVAC (1) is anattenuated canarypox virus-based vector that was a plaque-clonedderivative of the licensed canarypox vaccine, Kanapox (Tartaglia et al.(1992) Virology 188:217; U.S. Pat. Nos. 5,505,941, 5,756,103 and5,833,975—all of which are incorporated herein by reference). ALVAC (1)has some general properties which are the same as some generalproperties of Kanapox. ALVAC-based recombinant viruses expressingextrinsic immunogens have also been demonstrated efficacious as vaccinevectors (Tartaglia et al, In AIDS Research Reviews (vol. 3) Koff W.,Wong-Staol F. and Kenedy R. C. (eds.), Marcel Dekker NY, pp. 361-378(1993a); Tartaglia, J. et al. (1993b) J. Virol. 67:2370). For instance,mice immunized with an ALVAC (1) recombinant expressing the rabies virusglycoprotein were protected from lethal challenge with rabies virus(Tartaglia, J. et al., (1992) supra) demonstrating the potential forALVAC (1) as a vaccine vector. ALVAC-based recombinants have also provenefficacious in dogs challenged with canine distemper virus (Taylor, J.et al. (1992) Virology 187:321) and rabies virus (Perkus, M. E. et al.,In Combined Vaccines and Simultaneous Administration: Current Issues andPerspective, Annals of the New York Academy of Sciences (1994)), in catschallenged with feline leukemia virus (Tartaglia, J. et al., (1993b)supra), and in horses challenged with equine influenza virus (Taylor, J.et al., In Proceedings of the Third International Symposium on AvianInfluenza, Univ. of Wisconsin—Madison, Madison, Wis., pp. 331-335(1993)).

ALVAC (2) is a second-generation ALVAC vector in which vacciniatranscription elements E3L and K3L have been inserted within the C6locus (U.S. Pat. No. 5,990,091, incorporated herein by reference). TheE3L encodes a protein capable of specifically binding to dsRNA. The K3LORF has significant homology to E1F-2. Within ALVAC (2) the E3L gene isunder the transcriptional control of its natural promoter, whereas K3Lhas been placed under the control of the early/late vaccine H6 promoter.The E3L and K3L genes act to inhibit PKR activity in cells infected withALVAC (II), allowing enhancement of the level and persistence of foreigngene expression.

Additional viral vectors encompass natural host-restricted poxviruses.Fowlpox virus (FPV) is the prototypic virus of the Avipox genus of thePoxvirus family.

Replication of avipox viruses is limited to avian species (Matthews, R.E. F. (1982) Intervirology 17:42) and there are no reports in theliterature of avipox virus causing a productive infection in anynon-avian species including man. This host restriction provides aninherent safety barrier to transmission of the virus to other speciesand makes use of avipox virus based vectors in veterinary and humanapplications an attractive proposition.

FPV has been used advantageously as a vector expressing immunogens frompoultry pathogens. The hemagglutinin protein of a virulent avianinfluenza virus was expressed in an FPV recombinant. After inoculationof the recombinant into chickens and turkeys, an immune response wasinduced which was protective against either a homologous or aheterologous virulent influenza virus challenge (Taylor, J. et al.(1988) Vaccine 6: 504). FPV recombinants expressing the surfaceglycoproteins of Newcastle Disease Virus have also been developed(Taylor, J. et al. (1990) J. Virol 64:1441; Edbauer, C. et al. (1990)Virology 179:901); U.S. Pat. No. 5,766,599—incorporated herein byreference).

A highly attenuated strain of vaccinia, designated MVA, has also beenused as a vector for poxvirus-based vaccines. Use of MVA is described inU.S. Pat. No. 5,185,146. Other attenuated poxvirus vectors have beenprepared via genetic modification to wild type strains of vaccinia. TheNYVAC vector, for example, is derived by deletion of specific virulenceand host-range genes from the Copenhagen strain of vaccinia (Tartaglia,J. et al. (1992), supra; U.S. Pat. Nos. 5,364,773 and5,494,807—incorporated herein by reference) and has proven useful as arecombinant vector in eliciting a protective immune response againstexpressed foreign antigens.

Recombinant poxviruses can be constructed by a process known to thoseskilled in the art (as previously described for vaccinia and avipoxviruses; U.S. Pat. Nos. 4,769,330; 4,722,848; 4,603,112; 5,110,587; and5,174,993—all of which are incorporated herein by reference).

As such, one embodiment of the invention encompasses a recombinant viruscomprising a virus into which is inserted a nucleic acid encoding for apolypeptide of the invention, said recombinant virus causing theexpression of the polypeptide in an infected cell. In furtherembodiments of the invention the recombinant virus is selected from thegroup consisting of adenovirus, alphavirus and poxvirus. In stillfurther embodiments, the recombinant virus is ALVAC. In still yet afurther embodiment of the invention, the recombinant virus is ALVAC(1)into which is inserted the nucleic acid of FIG. 1 (SEQ ID NO:2) codingfor the polypeptide of FIG. 2 (SEQ ID NO:1). Additionally, therecombinant viral nucleic acid of said recombinant virus in oneembodiment of the invention is further characterized in that it isschematically represented in FIG. 3.

Bacterial DNA useful in embodiments of the invention have also beendisclosed and are known to those of ordinary skill in the art. Thesebacteria include, for example, Shigella, Salmonella, Vibrio cholerae,Lactobacillus, Bacille Calmette Guérin (BCG), and Streptococcus.

In further embodiments, live and/or attenuated bacteria per se may beused as vectors for nucleic acids of this invention. For example,non-toxicogenic Vibrio cholerae mutant strains may be useful asbacterial vectors in embodiments of this invention; as described in U.S.Pat. No. 4,882,278 (disclosing a strain in which a substantial amount ofthe coding sequence of each of the two ctxA alleles has been deleted sothat no functional cholera toxin is produced), WO 92/11354 (strain inwhich the irgA locus is inactivated by mutation; this mutation can becombined in a single strain with ctxA mutations), and WO 94/1533(deletion mutant lacking functional ctxA and attRS1 DNA sequences).These strains can be genetically engineered to express heterologousantigens, as described in WO 94/19482. (All of the aforementioned issuedpatent/patent applications are incorporated herein by reference.) Aneffective immunogen dose of a Vibrio cholerae strain capable ofexpressing a polypeptides encoded by a DNA molecule of the invention cancontain, for example, about 1×10⁵ to about 1×10⁹, preferably about 1×10⁶to about 1×10⁸ viable bacteria in an appropriate volume fox the selectedroute of administration.

Attenuated Salmonella typhimurium strains, genetically engineered forrecombinant expression of heterologous antigens and their use as oralimmunogens are described, for example, in WO 92/11361.

As previously noted, those skilled in the art will readily recognizethat other bacterial strains useful as sources of DNA and/or bacterialvectors in embodiments of this invention include Shigella flexneri,Streptococcus gordonii, and Bacille Calmette Guerin (as described in WO88/6626, WO 90/0594 WO 91/13157, WO 92/1796, and WO 92/21376; all ofwhich are incorporated herein by reference). In bacterial DNAembodiments of this invention, a nucleic acid of the invention may beinserted into the bacterial genome, can remain in a free state, or becarried on a plasmid.

Cells comprising the aforementioned nucleic acids coding forpolypeptides of this invention encompass further embodiments of theinvention. These cells encompass any potential cell into which a nucleicacid of this invention might be introduced and/or transfected. Thechoice of process tor the introduction and/or transfection into cells isdependant upon the intrinsic nature of the nucleic acid (i.e.recombinant virus, plasmid), as will be known to one skilled in the art(for example, as taught in Current Protocols in Molecular Biology, F. M.Ausubel et al. (Eds.), John Wiley and Sons, Inc., N.Y., U.S.A. (1998),Chpt. 9; Molecular Cloning: A Laboratory Manual (2^(nd) Ed.), J.Sambrook, E. F. Fritsch and T. Maniatis (Eds.), Cold Spring HarborLaboratory Press, N.Y., U.S.A. (1989), Chpts. 1, 2, 3 and 16).

It is well documented that the class I and class II proteins of themajor histocompatibility complex (MHC) perform a central immunologicalfunction in focusing T-lymphocytes of the immune system (i.e. CD8+ andCD4+ T lymphocytes). MHC class I proteins are expressed in nearly allnucleated cell types througout the human body; MHC class II moleculesare expressed mainly on antigen-presenting cells (APCs; namelymononuclear phagocytes, Langerhans-dendritic cells, and B lymphocytes).These distinct classes of cell surface molecules (i.e. class I and classII) present peptides/epitopes (derived from intracellular processing ofprotein antigens) to T lymphocytes (CD8+ and CD4+ T lymphocytesrespectively) thus initiating both cellular and humoral immuneresponses. Generally, epitopes/peptides derived from alloantigens, tumorantigens or viruses will be presented in association with MHC class Imolecules; extracellular antigens/proteins will be presented inassociation with MHC class II molecules. However, in some contextsendogenous antigens can also be presented in association with MHC classII molecules. [These general immunological principles are well describedin the art—as, for example, in Encyclopedia of Immunology (2^(nd) Ed.),Peter J. Delves (Ed.-in-Chief), Academic Press, San Diego, U.S.A., pp.174-8, 191-8, 1108-13, 1690-709 (1998).]

As such, embodiments of the invention encompass cells into which hasbeen introduced/transfected a nucleic acid of the invention, whereinsaid cells express a polypeptide of the invention. In furtherembodiments, said cells are capable of binding cleavage fragments (i.e.epitopes/peptides) of the polypeptide. In still yet further embodiments,these cleavage fragments are produced by a protease.

As conceived herein, embodiments of the invention may also encompasscells into which has been introduced/transfected a nucleic acid of theinvention wherein said cells also express a MHC HLA molecule (i.e. classI and/or class II). In further embodiments, these cells areantigen-presenting cells, possibly selected from the group consisting ofmononuclear phagocytes, Langerhans-dendritic cells (“dendriticcell(s)”), and B lymphocytes.

Additional embodiments of this invention further encompasspharmaceutical compositions comprising the aforementioned polypeptidesof the invention, nucleic acids coding therefor, and/or recombinantviruses comprising said nucleic acids for administration to subjects ina biologically compatible form suitable for administration in vivo. By“biologically compatible form suitable for administration in vivo” ismeant a form of the substance to be administered in which any toxiceffects are outweighed by the therapeutic effects. Administration of atherapeutically active amount of the pharmaceutical compositions of thepresent invention, or an “effective amount”, is defined as an amounteffective at dosages and for periods of time, necessary to achieve thedesired result of eliciting an immune response in a human. Atherapeutically effective amount of a substance may vary according tofactors such as the disease state/health, age, sex, and weight of therecipient, and the inherent ability of the particular polypeptide,nucleic acid coding therefor, or recombinant virus to elicit a desiredimmune response. Dosage regime may be adjusted to provide the optimumtherapeutic response. For example, several divided doses may beadministered daily or on at periodic intervals, and/or the dose may beproportionally reduced as indicated by the exigencies of the therapeuticsituation.

The compositions described herein can be prepared by per se knownmethods for the preparation of pharmaceutically acceptable compositionswhich can be administered to subjects, such that an effective quantityof the active substance (i.e. composition) is combined in a mixture witha pharmaceutically acceptable vehicle. Suitable vehicles are described,for example, in “Handbook of Pharmaceutical Additives” (compiled byMichael and Irene Ash, Gower Publishing Limited, Aldershot, England(1995)). On this basis, the compositions include, albeit notexclusively, solutions of the substances in association with one or morepharmaceutically acceptable vehicles or diluents, and may be containedin buffered solutions with a suitable pH and/or be iso-osmotic withphysiological fluids. In this regard, reference can be made to U.S. Pat.No. 5,843,456.

Methods of inducing or eliciting an immune response in an animaldirected against:

-   -   a polypeptide of the invention, or    -   a MAGE-specific antigen epitope of said polypeptide, or    -   a MAGE protein or fragment thereof comprising a MAGE-specific        antigen epitope, or    -   cells expressing a MAGE protein or fragment thereof, the        polypeptide of the invention, or a MAGE-specific antigen epitope        of the polypeptide, or    -   cells binding said MAGE protein or fragment thereof, the        polypeptide of the invention, or a MAGE-specific antigen epitope        of the polypeptide, comprising the step of administering to said        animal a polypeptide of the invention, a nucleic acid coding        therefor, a recombinant virus comprising said nucleic acid, or a        cell comprising said nucleic acid (or compositions of the        aforementioned) are also within the scope of this invention.        Further embodiments of this invention encompass treatments for        cancer comprising the aforementioned methods of inducing or        eliciting immune responses in animals.

As defined herein, a polypeptide of the invention, a nucleic acid codingtherefor, a recombinant virus comprising said nucleic acid, a cellcomprising said nucleic acid, and compositions of the aforementioned arecollectively referred to as “immunizing agent(s)”, “agent(s)”, or“immunogen(s)”.

As known to one of ordinary skill in the art, an animal may be immunizedwith a polypeptide of the invention, a nucleic acid coding therefor, arecombinant virus comprising said nucleic acid, or a cell comprisingsaid nucleic acid (or compositions of the aforementioned) by anyconventional route. This may include, for example, immunization via amucosal surface (e.g., ocular, intranasal, oral, gastric, pulmonary,intestinal, rectal, vaginal, or urinary tract) or via a parenteral route(e.g., subcutaneous, intradermal, intramuscular, intravenous, orintraperitoneal). Preferred routes depend upon the choice of theimmunogen (i.e. polypeptide vs. nucleic acid, composition formulation,etc.). The administration can be achieved in a single dose or repeatedat intervals. The appropriate dosage is dependant on various parametersunderstood by the skilled artisans, such as the immunogen itself (i.e.polypeptide vs. nucleic acid (and more specifically type thereof)), theroute of administration and the condition of the animal to be vaccinated(weight, age and the like). As such, embodiments of this inventionencompass methods of eliciting immune responses in animals comprisingadministering an effective amount of a polypeptide of the invention,nucleic acid coding therefore, recombinant virus comprising said nucleicacid, or cells comprising said nucleic acid (or compositions of theaforementioned).

As noted, nucleic acids (in particular plasmids and/or free/naked DNAand/or RNA coding for the polypeptide of the invention) can beadministered to an animal for purposes of inducing/exciting an immuneresponse (for example, U.S. Pat. No. 5,589,466; McDonnell and Askari,NEJM 334:42-45 (1996); Kowalczyk and Ertl, Cell Mol. Life Sci.55:751-770 (1999)). Typically, this nucleic acid is a form that isunable to replicate in the target animal's cell and unable to integratein said animal's genome. The DNA/RNA molecule encoding the polypeptideis also typically placed under the control of a promoter suitable forexpression in the animal's cell. The promoter can function ubiquitouslyor tissue-specifically. Examples of non-tissue specific promotersinclude the early Cytomegalovirus (CMV) promoter (described in U.S. Pat.No. 4,168,062) and the Rous Sarcoma Virus promoter. The desmin promoteris tissue-specific and drives expression in muscle cells. Moregenerally, useful vectors have been described (i.e., WO 94/21797).

For administration of nucleic acids coding for a polypeptide of theinvention, said nucleic acid can encode a precursor or mature form ofthe polypeptide. When it encodes a precursor form, the precursor formcan be homologous or heterologous. In the latter case, a eucaryoticleader sequence can be used, such as the leader sequence of thetissue-type plasminogen factor (tPA).

Standard techniques of molecular biology for preparing and purifyingnucleic acids well known to those skilled in the art can be used in thepreparation of aspects of the invention (for example, as taught inCurrent Protocols in Molecular Biology, F. M. Ausubel et al. (Eds.),John Wiley and Sons, Inc, N.Y., U.S.A. (1998), Chpts. 1, 2 and 4;Molecular Cloning: A Laboratory Manual (2^(nd) Ed.), J. Sambrook, E. F.Fritsch and T. Maniatis (Eds.), Cold Spring Harbor Laboratory Press,N.Y., U.S.A., (1989), Chpts. 1, 2, 3 and 7).

For use as an immunogen, a nucleic acid of the invention can beformulated according to various methods known to a skilled artisan.First, a nucleic acid can be used in a naked/free form, free of anydelivery vehicles (such as anionic liposomes, cationic lipids,microparticles, (e.g., gold microparticles), precipitating agents (e.g.,calcium phosphate) or any other transfection-facilitating agent. In thiscase the nucleic acid can be simply diluted in a physiologicallyacceptable solution (such as sterile saline or sterile buffered saline)with or without a carrier. When present, the carrier preferably isisotonic, hypotonic, or weakly hypertonic, and has a relatively lowionic strength (such as provided by a sucrose solution (e.g., a solutioncontaining 20% sucrose)).

Alternatively, a nucleic acid can be associated with agents that assistin cellular uptake. It can be, i.e., (i) complemented with a chemicalagent that modifies the cellular permeability (such as bupivacaine; see,for example, WO 94/16737), (ii) encapsulated into liposomes, or (iii)associated with cationic lipids or silica, gold, or tungstenmicroparticles.

Cationic lipids are well known in the art and are commonly used for genedelivery. Such lipids include Lipofectin (also known as DOTMA(N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP(1,2-bis(oleyloxy)-3-(trimethylammonio) propane). DDAB(dimethyldioctadecylammonium bromide), DOGS (dioctadecylamidologlycylspermine) and cholesterol derivatives such as DC-Chol (3beta-(N-(N′,N′-dimethyl aminomethane)carbamoyl) cholesterol). Adescription of these cationic lipids can be found in EP 187,702, WO90/11092, U.S. Pat. No. 5,283,185, WO 91/15501, WO 95/26356, and U.S.Pat. No. 5,527,928. Cationic lipids for gene delivery are preferablyused in association with a neutral lipid such as DOPE (dioleylphosphatidylethanolamine), as, for example, described in WO 90/11092.

Other transfection-facilitating compounds can be added to a formulationcontaining cationic liposomes. A number of them are described in, forexample, WO 93/18759, WO 93/19768, WO 94/25608, and WO 95/2397. Theyinclude, i.e., spermine derivatives useful for facilitating thetransport of DNA through the nuclear membrane (see, for example, WO93/18759) and membrane-permeabilizing compounds such as GALA,Gramicidine S, and cationic bile salts (see, for example, WO 93/19768).

Gold or tungsten microparticles can also be used for gene delivery (asdescribed in WO 91/359 and WO 93/17706). In this case, themicroparticle-coated polynucleotides can be injected via intradermal orintraepidermal routes using a needleless injection device (“gene gun”),such as those described, for example, in U.S. Pat. Nos. 4,945,050,5,015,580, and WO 94/24263.

Anionic and neutral liposomes are also well-known in the art (see, forexample, Liposomes: A Practical Approach, RFC New Ed, IRL Press (1990),for a detailed description of methods for making liposomes) and areuseful for delivering a large range of products, includingpolynucleotides.

As is well known to those of ordinary skill in the art, the ability ofan agent to induce/elicit an immune response can be improved if,regardless of administration formulation (i.e. recombinant virus,nucleic acid, polypeptide), said agent is co-administered with anadjuvant. Adjuvants are described and discussed in “Vaccine Design—theSubunit and Adjuvant Approach” (edited by Powell and Newman, PlenumPress, New York, U.S.A., pp. 61-79 and 141-228 (1995)). Adjuvantstypically enhance the immunogenicity of an immunogen but are notnecessarily immunogenic in and of themselves. Adjuvants may act byretaking the immunogen locally near the site of administration toproduce a depot effect facilitating a slow, sustained release ofimmunizing agent to cells of the immune system. Adjuvants can alsoattract cells of the immune system to an immunogen depot and stimulatesuch cells to elicit immune responses. As such, embodiments of thisinvention encompass compositions further comprising adjuvants.

Desirable characteristics of ideal adjuvants include:

-   -   1) lack of toxicity;    -   2) ability to stimulate a long-lasting immune response;    -   3) simplicity of manufacture and stability in long-term storage;    -   4) ability to elicit both cellular and humoral responses to        antigens administered by various routes, if required;    -   5) synergy with other adjuvants;    -   6) capability of selectively interacting with populations of        antigen presenting cells (APC);    -   7) ability to specifically elicit appropriate T_(H)1 or T_(H)2        cell-specific immune responses; and    -   8) ability to selectively increase appropriate antibody isotype        levels (for example, IgA) against antigens/immunogens.

However, many adjuvants are toxic and can cause undesirable sideeffects, thus making them unsuitable for use in humans and many animals.For example, some adjuvants may induce granulomas, acute and chronicinflammations (i.e. Freund's complete adjuvant (FCA)), cytolysis (i.e.saponins and pluronic polymers) and pyrogenicity, arthritis and anterioruveitis (i.e. muramyl dipeptide (MDP) and lipopolysaccharide (LPS)).Indeed, only aluminum hydroxide and aluminum phosphate (collectivelycommonly referred to as alum) are routinely used as adjuvants in humanand veterinary vaccines. The efficacy of alum in increasing antibodyresponses to diphtheria and tetanus toxoids is well established.Notwithstanding, it does have limitations. For example, alum isineffective for influenza vaccination and inconsistently elicits a cellmediated immune response with other immunogens. The antibodies elicitedby alum-adjuvanted antigens are mainly of the IgG1 isotype in the mouse,which may not be optimal for protection in vaccination contests.

Adjuvants may be characterized as “intrinsic” or “extrinsic”. Intrinsicadjuvants (such as lipopolysaccharides) are integral and normalcomponents of agents which in themselves are used as vaccines (i.e.killed or attenuated bacteria). Extrinsic adjuvants are typicallynonintegral immunomodulators generally linked to antigens in anoncovalent manner, and are formulated to enhance the host immuneresponse.

A variety of potent extrinsic adjuvants have been described. Theseinclude (but are not limited to) saponins complexed to membrane proteinantigens (immune stimulating complexes), pluronic polymers with mineraloil, killed mycobacteria and mineral oil, Freund's complete adjuvant,bacterial products such as muramyl dipepride (MDP) andlipopolysaccharide (LPS), as well as lipid A, and liposomes.

The use of saponins per se as adjuvants is also well known(Lacaille-Dubois, M. and Wagner, H. (1996) Phytomedicine 2:363). Forexample, Quil A (derived from the bark of the South American treeQuillaja Saponaria Molina) and fractions thereof has been extensivelydescribed (i.e. U.S. Pat. No. 5,057,540; Kensil, C. R. (1996) Crit RevTher Drug Carrier Syst. 12:1; and European Patent EP 362279). Thehaemolytic saponins QS21 and QS17 (HPLC purified fractions of Quil A)have been described as potent systemic adjuvants (U.S. Pat. No.5,057,540; European Patent EP 362279). Also described in thesereferences is the use of QS7 (a non-haemolytic fraction of Quil-A) whichacts as a potent adjuvant for systemic vaccines. Use of QS21 is furtherdescribed in Kensil et al. ((1991) J. Immunol 146:431). Combinations ofQS21 and polysorbate or cyclodextrin are also known (WO 9910008).Particulate adjuvant systems comprising fractions of Quil A (such asQS21 and QS7) are described in WO 9633739 and WO 9611711.

Another preferred adjuvant/immunostimulant is an immunostimulatoryoligonucleotide containing unmethylated CpG dinucleotides (“CpG”). CpGis an abbreviation for cytosine-guanosine dinucleotide motifs present inDNA. CpG is known in the art as being an adjuvant when administered byboth systemic and mucosal routes (WO 9602555; European Patent BP 468520;Davies et al. (1998) J. Immunol. 160:87; McCluskie and Davis (1998) J.Immunol 161:4463). In a number of studies, synthetic oligonucleotidesderived from BCG gene sequences have also been shown to be capable ofinducing immunostimulatory effects (both in vitro and in vivo; Krieg,(1995) Nature 374:546). Detailed analyses of immunostimulatoryoligonucleotide sequences has demonstrated that the CG motif must be ina certain sequence context, and that such sequences are common inbacterial DNA but are rare in vertebrate DNA. (For example, theimmunostimulatory sequence is often: purine, purine, C, G, pyrimidine,pyrimidine, wherein the CG motif is not methylated; however otherunmethylated CpG sequences are known to be immunostimulatory and as suchmay also be used in the present invention.)

A variety of other adjuvants are taught in the art, and as such areencompassed by embodiments of this invention. U.S. Pat. No. 4,855,283granted to Lockhoff et al. (incorporated herein by reference) teachesglycolipid analogues and their use as adjuvants. These includeN-glycosylamides, N-glycosylureas and N-glycosylcarbamates, each ofwhich is substituted in the sugar residue by an amino acid, asimmuno-modulators or adjuvants. Furthermore, Lockhoff et al. ((1991)Chem. Int. Ed. Engl. 30:1611) have reported that N-glycolipid analogsdisplaying structural similarities to the naturally-occurringglycolipids (such as glycophospholipids and glycoglycerolipids) are alsocapable of eliciting strong immune responses in both herpes simplexvirus vaccine and pseudorabies virus vaccine.

U.S. Pat. No. 4,258,029 granted to Moloney (incorporated herein byreference) teaches that octadecyl tyrosine hydrochloride (OTH) functionsas an adjuvant when complexed with tetanus toxoid and formalininactivated type I, II and III poliomyelitis virus vaccine. Nixon-Georgeet al. ((1990) J. Immunol. 14:4798) have also reported that octadecylesters of aromatic amino acids complexed with a recombinant hepatitis Bsurface antigen enhanced the host immune responses against hepatitis Bvirus.

Adjuvant compounds may also be chosen from the polymers of acrylic ormerhacrylic acid and the copolymers of maleic anhydride and alkenylderivative. Adjuvant compounds are the polymers of acrylic ormethacrylic acid which are cross-linked, especially with polyalkenylethers of sugars or polyalcohols. These compounds are known by the termcarbomer (Pharmeuropa Vol. 8, No, 2, June 1996). Preferably, a solutionof adjuvant according to the invention, especially of carbomer, isprepared in distilled water, preferably in the presence of sodiumchloride, the solution obtained being at acidic pH. This stock solutionis diluted by adding it to the desired quantity (for obtaining thedesired final concentration), or a substantial part thereof, of watercharged with NaCl, preferably physiological saline (NaCl 9 g/l) all atonce in several portions with concomitant or subsequent neutralization(pH 7.3 to 7.4), preferably with NaOH. This solution at physiological pHwill be used as it is for mixing with the immunizing agent; said mixturebeing amenable to storage in the freeze-dried, liquid or frozen form.

Persons skilled in the art can also refer to U.S. Pat. No. 2,909,462(incorporated herein by reference) which describes adjuvantsencompassing acrylic polymers cross-linked with a polyhydroxylatedcompound having at least 3 hydroxyl groups (preferably not more than 8),the hydrogen atoms of the at least three hydroxyls being replaced byunsaturated aliphatic radicals having at least 2 carbon atoms. Thepreferred radicals are those containing from 2 to 4 carbon atoms (e.g.vinyls, allyls and other ethylenically unsaturated groups). Theunsaturated radicals may themselves contain other substituents, such asmethyl. The products sold under the name Carbopol (BF Goodrich, Ohio,USA) are particularly appropriate. They are cross-linked with allylsucrose or with allyl pentaerythritol. Among them, there may bementioned Carbopol (for example, 974P, 934P and 971P). Among thecopolymers of maleic anhydride and alkenyl derivative, the copolymersEMA (Monsanto; which are copolymers of maleic anhydride and ethylene,linear or cross-linked, (for example cross-linked with divinyl ether))are preferred. Reference may be made to J. Fields et al, ((1960) Nature186: 778) for a further description of these chemicals (incorporated(herein by reference).

In further aspects of this invention, adjuvants useful for parenteraladministration of immunizing agent include aluminum compounds (such asaluminum hydroxide, aluminum phosphate, and aluminum hydroxy phosphate;but might also be a salt of calcium, iron or zinc, or may be aninsoluble suspension of acylated tyrosine, or acylated sugars,cationically or anionically derivatised polysaccharides, orpolyphosphazenes). The antigen can be precipitated with, or adsorbedonto, the aluminum compound according to standard protocols well knownto those skilled in the art.

Other adjuvants encompassed by embodiments of this invention includelipid A (in particular 3-de-O-acylated monophosphoryl lipid A (3D-MPL).3D-MPL is a well known adjuvant manufactured by Ribi Immunochem,Montana. Chemically it is often supplied as a mixture of 3-de-O-acylatedmonophosphoryl lipid A with 4, 5, or 6 acylated chains. It can beprepared by the methods taught in GB 2122204B. A preferred form of3D-MPL is in the form of a particulate formulation having a particlesize less than 0.2 μm in diameter (European Patent EP 689454).

Adjuvants for mucosal immunization may include bacterial toxins (e.g.,the cholera toxin (CT), the E. coli heat-labile toxin (LT), theClostridium difficile toxin A and the pertussis toxin (PT), orcombinations, subunits, toxoids, or mutants thereof). For example, apurified preparation of native cholera toxin subunit B (CTB) can be ofuse. Fragments, homologs, derivatives, and fusion to any of these toxinsare also suitable, provided that they retain adjuvant activity. A mutanthaving reduced toxicity may be used. Mutants have been described (e.g.,in WO 95/17211 (Arg-7-Lys CT mutant), WO 96/6627 (Arg-192-Gly LTmutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PT mutant)).Additional LT mutants include, for example Ser-63-Lys, Ala-69-Gly,Glu-110-Asp, and Glu-112-Asp mutants. Other adjuvants (such as abacterial monophosphoryl lipid A (MPLA)) of various sources (e.g., E.coli, Salmonella minnesota, Salmonella typhimurium, or Shigellaflexneri) can also be used in the mucosal administration of immunizingagents.

Adjuvants useful for both mucosal and parenteral immunization includepolyphosphate (for example, WO 95/2415), DC-choI (3 b-(N-(N′N′-dimethylaminomethane)-carbamoyl) cholesterol (for example, U.S. Pat. No.5,283,185 and WO 96/14831) and QS-21 (for example, WO 88/9336).

Adjuvants/immunostimulants as described herein may be formulatedtogether with carriers, such as for example liposomes, oil in wateremulsions, and/or metallic salts including aluminum salts (such asaluminum hydroxide). For example, 3D-MPL may be formulated with aluminumhydroxide (as discussed in EP 689454) or oil in water emulsions (asdiscussed in WO 9517210); QS21 may be advantageously formulated withcholesterol containing liposomes (as discussed in WO 9633739), in oilwater emulsions (as discussed in WO 9517210) or alum (as discussed in WO9815287). When formulated into vaccines, immunostimulatoryoligonucleotides (i.e. CpGs) are generally administered in free solutiontogether with free antigen (as discussed in WO 9602555; McCluskie andDavis (1998) Supra), covalently conjugated to an antigen (as discussedin WO 9816247), or formulated with a carrier such as aluminum hydroxideor alum (as discussed in Davies et al. Supra; Brazolot-Millan et al(1998) Proc. Natl. Acad. Sci. 95:15553).

Combinations of adjuvants/immunostimulants are also within the scope ofthis invention. For example, a combination of a monophosphoryl lipid Aand a saponin derivative (as described in WO 9400153, WO 9517210, WO9633739, WO 9856414, WO 9912565, WO 9911214) can be used, or moreparticularly the combination of QS21 and 3D-MPL (as described in WO9400153). A combination of an immunostimulatory oligonucleotide and asaponin (such as QS21), or a combination of monophosphoryl lipid A(preferably 3D-MPL) in combination with an aluminum salt also form apotent adjuvant for use in the present invention.

EXAMPLES

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitation.

Methods of molecular genetics, protein biochemistry and immunology usedbut not explicitly described in this disclosure and these Examples areamply reported in the scientific literature and are well within theability of those skilled in the art.

Example 1 Generation of the ALVAC-MAGE 1/3 Minigene RecombinantConstruct (vCP1469A)

Summary

To generate the ALVAC-MAGE 1/3 Minigene recombinant designated vCP1469A,a nucleic acid containing a poxvirus-promoted expression cassette for aminigene consisting of MAGE 1 and MAGE 3 epitopes separated by aputative protease cleavage site was subcloned into an ALVAC donorplasmid. This expression cassette was subsequently inserted into the C6insertion site in the ALVAC(1) genome by in vitro recombination.

No function has yet been ascribed to the C6 encoded polypeptide ofALVAC, nor does the deduced amino acid open reading frame encoded inthis region share significant homology to any entry in the existingprotein sequence databases. A schematic of the Xho I restriction mapprofile of the ALVAC(1)-MAGE 1/3 Minigene recombinant constructdesignated vCP1469A is shown in FIG. 3.

Generation of the vCP1469A ALVAC Recombinant

Plasmid pCDSRα(containing a sequence coding for a fragment of MAGE 1encompassing amino acids 149-181) was used as a template in a PCRreaction with the following primers:

MAGE01 (5′>CCC TCG CGA TAT CCG TTA AGT TTG TAT CGT AATGGA GTC CTT GCA GCT GGT C<3′), (SEQ ID NO.: 8), and MAGE02(5′>GGG CTC GAG CTA GTA CAA GTG GCC GAT GGG GTCCAG TTC TTT ACG CTT ATT GCC ATC ATA GGA GAG ACC TAG<3′) (SEQ ID NO.: 9).(This latter primer (i.e. MAGE02) comprises a nucleic acid antisensesequence for the MAGE 3-specific antigen epitope, proteolytic cleavagesite, and the 6 terminal amino acids at the carboxyl end of theabovenoted fragment of MAGE 1.)

The resulting 180 bp PCR fragment contained the 3′ end of an H6 promoter(originating from plasmid pCDSRα) linked to a MAGE 1/3 fusion gene. TheMAGE 1/3 fusion gene comprises the MAGE-1 specific antigen epitope(located within the MAGE-1 protein fragment consisting of amino acids149-181), an engineered protease cleavage site and the MAGE-3 specificantigen epitope (amino acids 161-169 of MAGE-3; the sequence of theselatter two provided by primer MAGE02 (as described above)). Thisfragment was digested with EcoR5 and Xho I and ligated with EcoR5/Xho Idigested plasmid pC6H6B7 (this latter plasmid contains an irrelevantH6-promoted coding sequence in a C6 donor plasmid). The resulting C6donor plasmid (designated pMAGE1/3-1) contains a regenerated H6 promoterlinked to the MAGE 1/3 Minigene.

DNA sequence analysis revealed a G to C substitution at position 118 ofthe fragment. To correct this nucleotide substitution, pMAGE 1/3-1 wasused as a template for PCR amplification utilizing primers MAGE03(5′>ATC GCG ATA TCC GTT AAG TTT G<3′) (SEQ) ID NO.: 10) and MAGE04(5′>GGG CTC GAG CTA GTA CAA GTG GCC GAT GGG GTC CAC TTC TTT ACG CTT ATTGCC<3′) (SEQ ID NO.: 11). The resulting 180 bp fragment containing the(3′) H6/MAGE-1/3 Minigene with corrected position 118 was digested withEcoR5 and Xho I and subsequently ligated with EcoR5/Xho I digested pMAGE1/3-1 vector. The resulting C6 donor plasmid (designated pC6MAGE 1/3CTL) contained the regenerated H6 promoter linked to the corrected MAGE1/3 Minigene.

Recombination was preformed between donor plasmid pC6MAGE 1/3 CTL andALVAC(1) rescuing virus utilizing procedures described in the art andknown to skilled artisans (i.e. U.S. Pat. Nos. 4,769,330, 4,722,848,4,603,112, 5,174,993, 5,110,587—all of which are incorporated herein byreference). The resultant recombinant virus (designated vCP1469A)comprises a vaccinia H6 promoted human MAGE 1/3 Minigene sequence in theC6 locus of ALVAC(1). It is characterized in that it comprises thenucleic acid insert whose sequence is depicted in FIG. 1 (SEQ ID NO:2)which codes for the polypeptide depicted in FIG. 2 (SEQ ID NO:1), and bythe Xho I restriction map profile depicted in FIG. 3.

Verification of Insertion

Viral genomic DNA was isolated from cells infected with vCP1469Apursuant to methods well known to those skilled in the art (for example,as taught in Current Protocols in Molecular Biology, F. M. Ausubel etal, (Eds.), John Wiley and Sons, Inc., N.Y., U.S.A. (1998); MolecularCloning: A Laboratory Manual (2* Ed.), J. Sambrook, E. F. Fritsch and T.Maniatis (Eds.), Cold Spring Harbor Laboratory Press, N.Y., U.S.A.(1989)). The genomic DNA was digested with restriction endonuclease XhoI. The resultant DNA fragments were fractionated by electrophoresisthrough an agarose gel and visualized by ethidium bromide staining. Theinsertion of the MAGE 1/3 Minigene expression cassette at the C6 locuswas confirmed (see FIG. 3 for a schematic representation, of therestriction map profile).

Example 2 Presentation of the Individual MAGE-Specific Antigen Epitopesof the MAGE1/3 Minigene In Suitable Target Cells

The capacity of the ALVAC-MAGE 1/3 Minigene recombinant virus (i.e.vCP1469A) to express and appropriately present the MAGE-1 and MAGE-3specific antigen epitopes in target cells has been analyzed using an invitro assay system.

Human dendritic cells (DCs) were isolated from peripheral bloodmononuclear cells via the culturing of non-adherent cells for 8 days inthe presence of GM-CSF and IL4. Cells were harvested and incubated for 6hours with recombinant virus (vCP1469A) at various multiplicities ofinfection (MOI; ranging from 0.9 to 60). Infected cells were washed andsubsequently tested for their capacity to stimulate IFN-gamma release byeither the CTL clone MZ2-CTL 82/30 (MAGE 1 specific, HLA-A1 restricted),or MZ2-CTL 20/38 (MAGE 3 specific, HLA-A1 restricted).

As depicted in FIG. 4, infected dendritic cells were able to inducesignificant production of IFN-gamma by both CTL clones, thusestablishing that infection with the recombinant virus resulted in theprocessing and presentation of both the MAGE-1 and MAGE-3 specificantigen epitope. Uninfected dendritic cells did not induce theproduction of detectable IFN-gamma.

Example 3 Presentation of the MAGE-1 Specific Antigen Epitope DerivedFrom the MAGE 1/3 Minigene In Association With Both MHC HLA-A1 AndHLA-B35 Molecules

Dendritic cells (DCs) of an HLA-A1 and B35 donor were infected withthree different ALVAC recombinant viruses. The first virus contained theMAGE 1/3 Minigene (i.e. vCP1469A); the second contained the codingsequence of MAGE-1 (designated MAGE-A1); the third contained the codingsequence of β-galactosidase (designated βgal; negative control). DCswere distributed in microwells (10⁴ cells per well), infected at variousmultiplicities of infection (MOI) for 2 hours, and subsequently washed.Melanoma cell lines MZ2-MEL.43 (HLA-A1⁺, HLA-B35⁻, MAGE-1⁺) andMI13443-MEL (HLA-A1⁺, HLA-B35⁺, MAGE-1⁺) were utilized as positivecontrol target cells. Three thousand cells of CTL clone MZ2-CTL 82/30(directed against the MAGE-1 epitope presented by HLA-A1) or CTL 7(directed against the MAGE-1 epitope presented by HLA-B35) were added tothe DCs or the positive control target cells. After 20 hours, IFN-γproduced by each CTL (indicative of an interaction between CTLs andtarget cells) was measured by ELISA.

As depicted in FIG. 5, infected DCs efficiently stimulated both relevantCTLs. The recognition of the epitope by CTL 7 was restricted by HLA-B35molecules; the recognition of the epitope by MZ2-CTL 82/30 wasrestricted by HLA-A1. These results indicate that the MAGE-1 specificantigen epitope of vCP1469A is presented in association with both HLA-A1and HLA-B35.

Example 4 Presentation of the MAGE-3 Specific Antigen Epitope DerivedFrom the MAGE 1/3 Minigene In Association With MHC HLA-B35 Molecules

Dendritic cells (DCs) of a B35 donor were infected with two differentALVAC recombinant viruses. The first virus contained the MAGE 1/3Minigene (i.e. vCP1469A); the second contained the coding sequence ofβ-galactosidase (designated βgal; negative control). DCs weredistributed in microwells (10⁴ cells per well), infected at variousmultiplicities of infection (MOI) for 2 hours, and subsequently washed.The melanoma cell line MI13443-MEL (HLA-A1⁺, HLA-B35⁺, MAGE-1⁺) wasutilized as a source of positive control target cells. Three thousandcells of CTL clone 41 (directed against the MAGE-3 epitope presented byHLA-B35) was added to the DCs or the positive control target cells.After 20 hours, IFN-γ produced by each CTL (indicative of an interactionbetween CTLs and target cells) was measured by ELISA.

As depicted in FIG. 6, infected DCs efficiently stimulated CTL clone 41.These results indicate that the MAGE-3 specific antigen epitope ofvCP1469A is presented in association with HLA-B35.

Example 5 Generation of A Clinical Response In A Human With Melanoma InResponse To the Administration of An ALVAC-MAGE 1/3 Minigene Recombinant(vCP1469A)

The administration of the ALVAC(1)-MAGE 1/3 Minigene recombinant(vCP1469A) to a subject with melanoma (i.e. multiple cutaneousin-transit metastases of the right leg) encompassed a “prime-boost”methodology.

“Priming” involved 4 sequential injections of the aforementioned ALVACrecombinant at 3-week intervals; “boosting” involved 3 sequentialinjections of MAGE 1 and 3 specific antigen epitope peptides (i.e.,EADPTGHSY (SEQ ID NO. 5) and EVDPIGHLY (SEQ ID NO. 6) respectively) at3-week intervals commencing 3 weeks after the last injection with ALVACrecombinant.

In respect of ALVAC recombinant injections (i.e. priming), 1 ml of ALVACrecombinant (10^(7.09)CCID₅₀) was divided between 4 injection sites—0.4ml was injected subcutaneously at sites 1 and 3, and 0.1 ml injectedinfradermally at sites 2 and 4 (into the deltoid region or the anterioraspect of the thighs). No injection was given into limbs whereindraining lymph nodes were surgically removed and/or irradiated, or inlimbs in which draining lymph nodes were known to contain metastases.

In respect of the MAGE-specific antigen epitope peptide injections (i.e.boosting), 0.5 ml of each peptide was divided between 2 separateinjection sites—0.4 ml was injected subcutaneously at sites 1 and 3, and0.1 ml injected infradermally at sites 2 and 4 (i.e. the boostingregiment with the 2 peptides consisted of 4 injection sites into thedeltoid or anterior aspect of the thigh).

The recipient of the ejections was routinely observed for a period of120 minutes immediately following any injection. During theseobservation periods, arterial blood pressure (systolic and diastolic),heart rate and body oral temperature were measured every 30 minutes. Therecipient was assessed every three weeks (with respect to history,physical examination, and basic biological tests). Color photographswere taken of all cutaneous and/or superficial lesions.

At the onset of the clinical trial, the recipient of the injectionsmanifested melanoma in the form of multiple cutaneous in-transitmetastases of the right leg. After receiving four series of injectionswith the ALVAC recombinant and 2 series of injections with the MAGEpeptides, an objective regression of cutaneous metastases was observed.A minority of modules demonstrated some regression during the initialseries of “priming” injections with the ALVAC recombinant. No newlesions have appeared, and bleeding lesions have resorbed. Most lesionsdecreased in size (mostly in depth); larger lesions became necrotic andgradually shrank. An enlargement of a right inguinal lymph nodedeveloped. An examination of said lymph node did not reveal the presenceof melanoma cells.

Having illustrated and described the principles of the invention in apreferred embodiment, it should be appreciated by those skilled in theart that the invention can be modified in arrangement and detail withoutdeparture from such principles. We claim all modifications coming withinthe scope of the following claims. All publications, patents and patentapplication referred to herein, are herein incorporated by reference intheir entirety to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto the incorporated by reference in its entirety.

1. A method of inducing an immune response against a MAGE protein in ananimal comprising administering to the animal a nucleic acid moleculeencoding a polypeptide comprising a first amino acid sequence Glu SerLeu Gln Leu Val Phe Gly Ile Asp Val Lys Glu Ala Asp Pro Thr Gly His SerTyr Val Leu Val Thr Cys Leu Gly Leu Ser Tyr Asp Gly (amino acid residues2-34 of SEQ ID NO: 1) and a second amino acid sequence Glu Val Asp ProIle Gly His Leu Tyr (SEQ ID NO: 6).
 2. The method of claim 1 wherein thefirst and second amino acid sequences are separated by a proteasecleavage site.
 3. The method of claim 2 wherein the protease cleavagesite has the amino acid sequence Asn Lys Arg Lys (amino acid residues35-38 of SEQ ID NO: 1).
 4. The method of claim 1 wherein the first aminoacid sequence is Met Glu Ser Leu Gln Leu Val Phe Gly Ile Asp Val Lys GluAla Asp Pro Thr Gly His Ser Tyr Val Leu Val Thr Cys Leu Gly Leu Ser TyrAsp Gly (amino acid residues 1-34 of SEQ ID NO: 1).
 5. The method ofclaim 4 wherein the protease cleavage site has the amino acid sequenceAsn Lys Arg Lys (amino acid residues 35-38 of SEQ ID NO: 1).
 6. Themethod of claim 1 wherein the nucleic acid molecule Comprises SEQ ID NO:2.
 7. The method of claim 1 wherein the polypeptide comprises SEQ IDNO:
 1. 8. A method of inducing an immune response against a MAGE proteinin an animal comprising administering to the animal a recombinantexpression vector comprising a nucleic acid molecule encoding apolypeptide comprising a first amino acid sequence Glu Ser Leu Gln LeuVal Phe Gly Ile Asp Val Lys Glu Ala Asp Pro Thr Gly His Ser Tyr Val LeuVal Thr Cys Leu Gly Leu Ser Tyr Asp Gly (amino acid residues 2-34 of SEQID NO: 1) and a second amino acid sequence Glu Val Asp Pro Ile Gly HisLeu Tyr (SEQ ID NO: 6).
 9. The method of claim 8 wherein the expressionvector is selected from the group consisting of a, plasmid, and a viralvector.
 10. The method of claim 9 wherein the viral vector is selectedfrom the group consisting of adenovirus, alphavirus and poxvirus. 11.The method of claim 10 wherein the poxvirus is ALVAC or ALVAC(2). 12.The method of claim 1 wherein the nucleic acid molecule is administeredas part of a composition comprising a pharmaceutically acceptablecarrier.
 13. The method of claim 8 wherein the recombinant expressionvector is administered as part of a composition comprising apharmaceutically acceptable carrier.
 14. The method of claim 1 whereinthe animal is a human being.
 15. The method of claim 8 wherein theanimal is a human being.
 16. The method of claim 12 wherein the animalis a human being.
 17. The method of claim 13 wherein the animal is ahuman being.